CN103614393A - CYP2D6 gene segment containing 1678T>C mutation, coded protein fragment thereby and applications thereof - Google Patents
CYP2D6 gene segment containing 1678T>C mutation, coded protein fragment thereby and applications thereof Download PDFInfo
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Abstract
The invention belongs to the biological field, and relates to single base mutation of the 1678th locus of CYP2D6 allele. The locus is mutated into C from T. The invention concretely relates to a nucleic acid segment containing the mutation locus, a corresponding coded protein fragment, agents for identification of the mutation locus, a detection method and applications of the locus, especially applications of the identification of the locus in medication guidance.
Description
Technical field
The invention belongs to field of biology, relate to the single base mutation of the 1678th, CYP2D6 allelotrope.More specifically, the nucleic acid fragment that the present invention relates to comprise this mutational site and the protein fragments of corresponding encoded thereof, the reagent of identifying described mutational site, the application in direction of medication usage of detection method and this site of evaluation.
Background technology
Cytochrome P 4502 D 6 (CYP2D6) is one of important member of CYP enzyme family.Its gene is positioned on No. 22 karyomit(e), comprises 9 exons, full length gene 9432bp (GenBank number of registration M33388, exon 1 is positioned at 1620-5909 position).In human body, the amount of these cytopigment only accounts for 2%～4% of liver enzyme total amount, but participate in the metabolism of 20%～30% medicine clinically, medicine comprises thymoleptic, anti-arrhythmic, antipsychotic drug, anodyne, antitussive, antiemetic, antidiabetic drug and Bextra etc. like this, studies its metabolic polymorphism and has very important clinical value (referring to reference 1).
CYP2D6 gene has height polymorphism.So far, in NCBI snp database, included and surpassed 300 mutational sites.Allelotrope by the international name of CYP450 committee member name also has more than 150, also has in addition multiple newfound saltant type not yet by name (http://www.cypalleles.ki.se/cyp2d6.htm).Each allelotrope relates to point mutation, disappearance, insertion, rearrangement etc. in various degree, thereby affects the activity of CYP2D6, and then the metabolic activity of impact to medicine, produces drug effect in various degree.Remove outside wild-type CYP2D6*1, at present research saltant type more and that clinical meaning is larger mainly comprises following 7 kinds: CYP2D6*2, * 3, * 4, * 5, * 10, * 17, * 41, wherein ethnic group distribute the widest, most study, Chinese population research data relatively the abundantest saltant type be CYP2D6*2 (2850C > T; 4180G > C) and CYP2D6*10 (100C > T; 4180G > C) (referring to reference 1,2,3).
According to current clinical studies show, this polymorphism of CYP2D6 gene is to cause CYP2D6 enzymic activity in the significant major cause of interindividual variation, carry the very big difference that can cause curative effect of medication between the genotypic individuality of different CYP2D6, even produce serious poisonous side effect of medicine or treat insufficient.Therefore, research CYP2D6 gene pleiomorphism will provide important scientific basis (referring to reference 3,4) to the impact of curative effect of medication to clinical rational drug use.
Summary of the invention
The new single base mutation site that the object of this invention is to provide CYP2D6 gene, the nucleic acid fragment that comprises this mutational site, the protein fragments of its coding and identify the application of this mutational site in medication guide.
First aspect of the present invention is to provide nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1678th corresponding to SEQ IDNO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, wherein the Nucleotide of the 1678th is C; Or described nucleic acid fragment comprises the mutational site of the 425th corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 425th is C; Or the reverse complementary sequence that described nucleic acid fragment is above-mentioned nucleic acid fragment.
Second aspect of the present invention is to provide and contains corresponding to the 1678th of SEQ ID NO.1 or corresponding to the allelotrope fragment in the mutational site of the 425th or the allele specific oligonucleotide of all or part of hybridization of its reverse complementary sequence of SEQ ID NO.2, and wherein the Nucleotide in the mutational site of the 425th of the 1678th of SEQ ID NO.1 the or SEQ ID NO.2 is C; Described allelotrope fragment is at least 10 continuous nucleotides or its reverse complementary sequence in the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
The 3rd aspect of the present invention is to provide for detection of and/or analyzes the test kit of single base mutation of the present invention, described test kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprise can be as single base mutation described in primer amplification but do not comprise the nucleic acid fragment of this list base; Described single base is corresponding to the 1678th or the 425th of SEQ ID NO.2 of SEQ ID NO.1.
The 4th aspect of the present invention is to provide nucleic acid fragment of the present invention or the application of oligonucleotide in detecting CYP2D6 transgenation, and wherein said nucleic acid fragment or oligonucleotide are as probe or primer; Or nucleic acid fragment of the present invention or oligonucleotide are for the preparation of the application that detects the medicine of CYP2D6 transgenation; Or nucleic acid fragment of the present invention or oligonucleotide are as the application that detects the check mark thing of CYP2D6 transgenation.
The 5th aspect of the present invention is to provide a kind of medication guide, comprises the 1678th or the single base mutation of the 425th of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2D6 gene in testing sample; According to the sudden change detecting, adjust the dosage by the medicine of CYP2D6 metabolism.
The 6th aspect of the present invention is to provide the method for analysis of nucleic acids, described method comprise analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1678th or analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the Nucleotide of the 425th.
The 7th aspect of the present invention is to provide CYP2D6 albumen or its fragment or varient, and described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the Serine of the 142nd corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3.
The invention provides the CYP2D6 gene and the encoding sequence that comprise new single base mutation.This gene sports C (425T > C) at the 425th Nucleotide corresponding to SEQ ID NO.2 by T, thereby the amino acid that causes its coding sports Serine by leucine, corresponding to the Serine of the 142nd of SEQ ID NO.3.The CYP2D6 albumen of this sudden change (called after L142S) is compared reduction to the metabolic activity of medicine with wild-type.This single base mutation has directive significance to carrying the individual medication in this mutational site.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates that checks order of the heterozygote carrier corresponding to SEQ ID NO.1 sequence of the present invention in embodiment 1, and wherein arrow indication is the 1678th Nucleotide of CYP2D6 allelotrope;
Fig. 2 is the structural representation collection of illustrative plates of the double gene expression vector pIRES-Gluc-2D6 of embodiment 2 structures;
Fig. 3 be the CYP2D6.1 shown in embodiment 2 (wild-type), CYP2D6.2 (deficient mutants), CYP2D6.10 (deficient mutants) and L142S albumen of the present invention for the metabolic capacity detected result of Dextromethorphane Hbr and bufuralol, wherein * represents p value < 0.05;
Fig. 4 is that the CYP2D6.1 shown in embodiment 2 (wild-type), CYP2D6.2 (deficient mutants), CYP2D6.10 (deficient mutants) and L142S albumen of the present invention are for the typical LC-MS/MS detection collection of illustrative plates of Dextromethorphane Hbr metabolism; Arrow indication place is respectively the fignal center of substrate Dextromethorphane Hbr (right side) and meta-bolites demethyl Dextromethorphane Hbr (left side) thereof; Wherein X-coordinate represents retention time, and the retention time of Dextromethorphane Hbr and demethyl Dextromethorphane Hbr is respectively 7.5 and 4.8 minutes, ordinate zou expression signal response value cps.
Fig. 5 is that the CYP2D6.1 shown in embodiment 2 (wild-type), CYP2D6.2 (deficient mutants), CYP2D6.10 (deficient mutants) and L142S albumen of the present invention are for the typical LC-MS/MS detection collection of illustrative plates of bufuralol metabolism; Arrow indication place is respectively the fignal center of substrate bufuralol (right side) and meta-bolites 1 '-hydroxyl bufuralol (left side) thereof; Wherein X-coordinate represents retention time, and the retention time of bufuralol and 1 '-hydroxyl bufuralol is respectively 6.9 and 5.1 minutes, ordinate zou expression signal response value cps.
Embodiment
By following embodiment explanation the present invention, but content of the present invention is not limited to this.
As illustrated without other, " nucleic acid fragment " of the present invention is comprised of Nucleotide or its analogue, can be the fragment of DNA, RNA or its analogue; Can be strand or two strands; It can be natural (as genomic) or synthetic.
In the present invention, " sudden change " refers at the gene detecting, in CYP2D6 gene, has the nucleotide site different from wild-type CYP2D6 gene order." mutational site " refers to the position that base is undergone mutation.In the present invention, described mutational site is corresponding to the 425th in sequence shown in the 1678th of sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
International P450 allelotrope NK is about stipulating in CYP2D6 allelotrope naming rule: using first base A of initiator codon ATG in CYP2D6 genomic dna reference sequences (GenBank number of registration M33388) as allelic the 1st of CYP2D6 (first base A of initiator codon is positioned at the 1620th of M33388 sequence), the definite catastrophe point of the present invention is positioned at allelic the 1678th of CYP2D6 (SEQ ID NO.1).
Content of the present invention relates to the nonsynonymous mutation of CYP2D6 gene.Because this mutational site is arranged in the encoding sequence of gene, therefore, those skilled in the art are known, and described mutational site both can show in genomic dna, also can in encoding sequence (being CDS), show.Those skilled in the art, according to detected sample, can detect this mutational site in genomic dna or mRNA level.In the application, SEQ ID NO.1 is that wherein the 1678th is the mutational site the present invention relates to according to the CYP2D6 allelotrope sequence of the present invention of the stipulative definition of international P450 allelotrope NK.SEQ ID NO.2 is the cDNA sequence with the CYP2D6 gene in described mutational site, and wherein the 425th is the mutational site the present invention relates to.Those skilled in the art are known, in this article, and corresponding to the 1678th site of SEQ ID NO.1 with use mutually corresponding to the 425th the site synonym of SEQ ID NO.2.
In the present invention, " allele-specific " refer to specifically and allelotrope hybridization, as hybridized under rigorous condition, makes to identify that the 425th Nucleotide corresponding to sequence shown in the 1678th of sequence shown in SEQ ID NO.1 or SEQ IDNO.2 is C.
In the present invention, Nucleotide and amino acid whose abbreviation adopt abbreviation mode well known in the art, and as A in Nucleotide represents VITAMIN B4, G represents guanine, and C represents cytosine(Cyt), and T represents thymus pyrimidine.In amino acid, A represents L-Ala, and R represents arginine, and N represents l-asparagine, D represents aspartic acid, and C represents halfcystine, and Q represents glutamine, and E represents L-glutamic acid, G represents glycine, and H represents Histidine, and I represents Isoleucine, and L represents leucine, K represents Methionin, and M represents methionine(Met), and F represents phenylalanine, P represents proline(Pro), and S represents Serine, and T represents Threonine, W represents tryptophane, and Y represents tyrosine, and V represents α-amino-isovaleric acid.
Content of the present invention is the new single base mutation site based on CYP2D6 gene.Described mutational site is the coding region that is positioned at CYP2D6 gene, and corresponding to the 425th of SEQ ID NO.2, this site sports C by the T of wild-type; In addition, the 142nd by the albumen of the CYP2D6 genes encoding of this sudden change sports Serine (L142S) by leucine.
Aspect first, the invention provides nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1678th corresponding to SEQ IDNO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, and wherein the Nucleotide of the 1678th is C; Or described nucleic acid fragment comprises the mutational site of the 425th corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 425th is C; Or be the reverse complementary sequence of above-mentioned nucleic acid fragment.
In one embodiment, the length of described nucleic acid fragment can be as 10-100,101-200, a 201-500 or 501-1000 Nucleotide.Preferably, the length of described nucleic acid fragment is 10-20,21-30,31-40,41-50,51-60, a 61-100 or 101-300 Nucleotide.
Described mutational site can be positioned at any position of described nucleic acid fragment.
In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.1.
In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.2.
In other embodiments, described nucleic acid fragment can be the sequence shown in SEQ ID NO.14-18.
Second aspect of the present invention is to provide and contains corresponding to the 1678th of SEQ ID NO.1 or corresponding to the allelotrope fragment in the mutational site of the 425th or the allele specific oligonucleotide of all or part of hybridization of its reverse complementary sequence of SEQ ID NO.2, and wherein the Nucleotide in the mutational site of the 425th of the 1678th of SEQ ID NO.1 the or SEQ ID NO.2 is C; Described allelotrope fragment is at least 10 continuous nucleotides or its reverse complementary sequence in the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
In one embodiment, described oligonucleotide is as probe.Described probe can be under rigorous condition and the target sequence specific hybrid that comprises mutational site.It is known to those skilled in the art that described probe does not need and target sequence complete complementary, if can with target sequence specific hybridization.In preferred embodiments, described hybridization conditions can meet make probe only with target sequence specific hybrid.The length of described probe can be 5-100 Nucleotide, as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,40,50,60,70,80,90 or 100 Nucleotide.Described mutational site can appear at any position of probe.In a preferred embodiment, described mutational site appears at center or about center of probe sequence.
In another embodiment, described oligonucleotide is with coaching the synthetic primer of DNA, as known in the art sequencing primer or synthetic primer etc.Described primer does not need and template complete complementary, but should be with the complementary hybridization of template to instruct DNA synthetic.The length of described primer can be 15-40 length of nucleotides, is preferably 18,19,20,21,22,23,24,25,26,27,28,29 or 30 Nucleotide.Described mutational site can appear at any position of described primer; Preferably, described mutational site appears at 3 ' end of described primer.
Some preferred embodiment in, described oligonucleotide is the sequence as shown in SEQ ID NO.19-23.
Based on this, the 3rd aspect of the present invention be to provide for detection of and/or the test kit of analysis list base mutation, described test kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprise can be as single base mutation described in primer amplification but do not comprise the nucleic acid fragment of this list base; Described single base is corresponding to the 1678th or the 425th of SEQ ID NO.2 of SEQ ID NO.1.Preferably, described test kit comprises the sequence fragment shown in SEQ ID NO.4 and/or SEQ ID NO.5 and/or SEQ ID NO.10.
The 4th aspect of the present invention is to provide nucleic acid fragment of the present invention or oligonucleotide for detection of the application of CYP2D6 transgenation, and wherein said nucleic acid fragment or oligonucleotide are as probe or primer; Or nucleic acid fragment of the present invention or oligonucleotide are for the preparation of the application that detects the medicine of CYP2D6 transgenation; Or nucleic acid fragment of the present invention or oligonucleotide are as the application that detects the check mark thing of CYP2D6 transgenation.
The 5th aspect of the present invention is to provide medication guide, comprises the 1678th or the base of the 425th of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2D6 gene in testing sample.When the CYP2D6 gene detecting is C in the site of the 425th corresponding to the 1678th of SEQ ID NO.1 or SEQ ID NO.2, adjust accordingly the dosage through the medicine of CYP2D6 metabolism.In specific embodiment, when CYP2D6 gene is C in the site of the 425th of SEQ ID NO.2, the CYP2D6 protease activity of this genes encoding declines, therefore need to adjust the dosage through the medicine of CYP2D6 metabolism, assigns as follows dose.
The medicine through CYP2D6 metabolism described in the present invention comprises: beta-blockers, Proprasylyte, metoprolol, H-56/28, bufuralol, timolol, bunitrolol, carvedilol, alprenolol, nebivolol; Anti-arrhythmic, encainide, sparteine, Tamboar, Propafenone, aprindine, mexiletine, encainide, procainamide; Antihypertensive drug, Debrisoquine, Indoramine; Antianginal, perhexiline, terodiline; Anodyne, U-26225A; Anti-spiritual medicine, chlorpromazine, trilafon, haloperidol, risperidone, thioridazine, zuclopenthixol, Aripiprazole; Tricyclic antidepressant, amitriptyline, imipramine, chlorimipramine, Desipramine, nortriptyline; Other thymoleptic, fluoxetine, paroxetine, Venlafaxine, fluvoxamine, amiflamine, mianserin, brofaromine, maprotiline, tomoxetine, amphetamine, citalopram, fluvoxamine, Minaprine, duloxetine, moclobemide; Medicine for the treatment of cough and asthma, dihydrocodeine, Ethylmorphine, morphine monomethyl ether, Dextromethorphane Hbr; Antidiabetic drug, phenformin; Other, Toldrin, metoclopramide, Tomoxetine hydrochloride, Isomeride, ondansetron, amphetamines, lignocaine, ondansetron, Phenacetin, triphen chloramines, dexfenfluramine, promethazine.
The 6th aspect of the present invention is to provide the method for analysis of nucleic acids, described method comprise analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1678th or analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the Nucleotide of the 425th.
In one embodiment, described method can be restriction fragment length polymorphism analysis (RFLP).Those skilled in the art can be according to the present invention the content design experiment Nucleotide of the 425th of take in the nucleic acid of sequence of the Nucleotide of the 1678th in the nucleic acid of the sequence of analyzing SEQ ID NO.1 or SEQ ID NO.2 whether be C.
In another embodiment, described method can be sequencing, comprise separated and measure the nucleotide sequence from genomic dna or RNA, analyze wherein comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1678th or whether comprise corresponding to the Nucleotide corresponding to the 425th in the nucleic acid of the sequence of SEQ ID NO.2 be C.Sequencing can be any available sequence measurement known in the art.Sequencing primer can design according to those skilled in the art's general knowledge, and the upstream and downstream appropriate position design primer as in site to be detected, contains the fragment in this site to be measured, thereby judge the Nucleotide in this site with expanding packet.Also can adopt oligonucleotide of the present invention as primer sequence.
In another embodiment, described method is to utilize the method for probe hybridization, in identification and detection sample, comprise specifically corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1678th or whether comprise corresponding to the Nucleotide corresponding to the 425th in the nucleic acid of the sequence of SEQ ID NO.2 be C; The probe adopting in described method is oligonucleotide of the present invention.For example, from testing sample, isolate nucleic acid, under allowing the condition of probe and the specific target sequence hybridization that may exist in nucleic acid, probe is contacted with nucleic acid; The hybridization that can be detected can be by using the probe of being crossed by detectable reagent mark to realize; For example,, with radio isotope, fluorescence dye or can catalysis form the enzyme that can detect product and carry out label probe.Label probe, with label probe, detect that in sample, whether to have the method for target sequence be all well-known to those skilled in the art.
In a kind of concrete embodiment, provide with Taqman probe SNP detection method and detect the method corresponding to the Nucleotide of the 1678th of SEQ ID NO.1, comprising:
1) design primer comprises the PCR product of the 1678th corresponding to SEQ ID NO.1 for specific amplification, designs two Taqman-MGB probes, respectively for T and the C allelotrope of the 1678th corresponding to SEQ ID NO.1 simultaneously.
Design of primers principle is:
(1) choose should be at the conservative section of gene for sequence;
(2) avoid primer self or and primer between form the above pairing continuously of 4 or 4, avoid primer self to form pili annulati card structure;
(3) primer length is at 18 to 24 Nucleotide;
(4) Tm value is at 55-65 ℃, and GC content is at 40%-60%;
(5) the Tm value between primer differs and avoids over 2 ℃;
(6) 3 ' of primer end avoids using base A, and 3 ' end of primer avoids occurring 3 or 3 above consecutive identical bases;
(7) pcr amplified fragment length is at 50bp-150bp;
(8) last 5 Nucleotide of primer end can not have G and the C that surpasses 2.
Taqman MGB probe design principle is:
(1) 5 ' of probe end avoids occurring G;
(2) Tm value should be 65-67 ℃;
(3) shorten Taqman MGB probe, but probe length is no less than 13bp as far as possible;
(4) avoid the base, especially the G base that duplicate as far as possible, avoid occurring that 4 or 4 above G repeat;
(5) mutational site of probe is placed on as far as possible to the place of centre 1/3.
Fluorophor can adopt FAM, VIC etc. to carry out two allelotrope of mark.
2) utilize above-mentioned primer and probe, sample to be tested is carried out to real-time quantitative PCR.
PCR condition: 95 ℃ of denaturations enter 30 amplification cycles after 10 minutes: 92 ℃ of sex change 12 seconds, 60 ℃ of annealing and extend 1 minute (this stage is detected fluorescent signal).
3) data analysis.
Analyze experimental result, according to the power of two kinds of fluorescence of sample, judge whether sample to be tested CYP2D6 gene exists 1678T > C sudden change.
In the present invention, described sample can be any sample that comprises nucleic acid, as blood; Preferred described sample comes from people.Described nucleic acid can be DNA or coding RNA, is preferably genomic dna.It is target compound that the method for analysis of nucleic acids of the present invention can be take DNA or RNA.Those skilled in the art are known, when take DNA when detecting target compound, analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1678th, the probe using or primer are according to the sequences Design of SEQ ID NO.1; When take RNA when detecting target compound, analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the Nucleotide of the 425th, the probe using or primer are according to the sequences Design of SEQ ID NO.2.
The 7th aspect of the present invention is to provide CYP2D6 albumen or its fragment or varient, and described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the Serine of the 142nd corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3, as 10-20,21-50 or 51-100 amino acid.
To further illustrate the present invention by specific embodiment below, but following specific embodiment is only for exemplary object.
Embodiment 1: the evaluation in the mutational site that people CYP2D6 gene is new
In the present embodiment, gather Normal Occlusion of Han People healthy population blood sample, extract the genomic dna in blood, design sequencing primer carries out sequence amplification, order-checking to 9 of CYP2D6 gene exons, analyzes its CYP2D6 gene and whether has mutational site.
1) extract DNA:
From measured, take 5ml vein EDTA anticoagulated blood sample; Then according to common salting-out process and/or adopt special DNA extraction test kit (purchased from the DNA extraction test kit of U.S. Omega company) to extract the genomic dna of blood sample to be measured.
2) pcr amplification:
Design of amplification primers, increases to 9 exon sequences of CYP2D6 gene in the genome DNA sample obtaining.Described amplimer to sequence in Table 1.
Adopt 30 μ L PCR reaction systems, comprising: 1 * GC PCR damping fluid, 1.5mM MgCl
2, the genomic dna of 100ng, upstream and downstream primer be the LATaq archaeal dna polymerase 1.5U that 0.2 μ M, dNTP are 0.2mM, TaKaRa company.Use the GeneAmp PCR System9700 amplification instrument amplification of American AB I company.Pcr amplification loop parameter is as follows: 94 ℃ of denaturations 2 minutes, and 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 68 ℃ are extended 3 minutes, after 35 circulations, extend 3 minutes again.Primer sequence information is in Table 1.
Table 1: amplimer is to sequence information
3) purifying amplified production:
By the amplified production obtaining according to MultiScreen HTS
tMthe operation instruction of test kit (U.S. Millipore company), the DNA that carries out object band reclaims purifying.
4) order-checking:
The product of take after reclaiming is template, use sequencing primer according to the PCR reaction of checking order of BigDye Terminator v3.1 sequencing kit (American AB I company) operation instruction, reaction finishes rear purifying amplified production, uses the Prism3730XL type gene sequencer of American AB I company to carry out the separated sequence with interpretation amplified production.Sequencing primer sequence information is in Table 2.
Table 2: sequencing primer sequence information table
| Region | Sequencing primer (5 '-3 ') |
| Exons 1 | AGGAAGCAGGGGCAAGAAC(SEQ?ID?NO.8) |
| Exon 2 | CGCCCTCTCTGCCCAGC(SEQ?ID?NO.9) |
| Wai Xianzi3 &4 | TTGGAGTGGGTGGTGGA(SEQ?ID?NO.10) |
| Exon 5&6 | AGGARGTYAGGCTTACAGGA(SEQ?ID?NO.11) |
[0085]?
| Exon 7 | GCACAGGCTTGACCAGGAT(SEQ?ID?NO.12) |
| Exon 8&9 | TGTTTGGTGGCAGGGGTCC(SEQ?ID?NO.13) |
5) data analysis:
The sequence recording and wild-type CYP2D6*1 sequence (GenBank number of registration M33388) are compared.
Pass through compare of analysis, find to have in 2129 routine experimenters 1 people's CYP2D6 genomic dna sequence to carry a kind of brand-new mutation type, the Nucleotide that corresponding CYP2D6 is allelic the 1678th becomes C (as shown in Figure 1, wherein Y represents T and C heterozygosis) from T.This sudden change is positioned at the 3rd exon of CYP2D6 gene, and catastrophe point is positioned at the 425th of cDNA, infers accordingly in the protein of this CYP2D6 genes encoding, and the 142nd amino acids sports Serine (S) by leucine (L).This sudden change Xian Yibei P450 called after neomorph CYP2D6*89 of NK, but not yet externally announce.
The present embodiment has exemplarily provided the method for identifying new mutant site.Those skilled in the art can clearly learn to detect specifically according to foregoing and in testing sample, comprise the method corresponding to the 1678th Nucleotide of SEQ ID NO.1: the nucleic acid in sample separation, under corresponding experiment condition, carry out amplified reaction in the present embodiment, primer is used primer pair SEQ ID NO.4 and 5; With sequencing primer SEQ ID NO.10, the product of amplification is checked order; Sequencing result and wild-type result are compared, analyzed the Nucleotide corresponding to the 1678th site of SEQ ID NO.1.
Embodiment 2: vitro enzyme metabolic activity is analyzed
According to existing result of study, wild-type (* 1 type) is all higher to the metabolic activity of various medicines, and the metabolic activity of * 2 types has obvious decline than the metabolic activity of wild-type, and the metabolic activity of * 10 types is than * 2 types lower (referring to document 7,8).Therefore, have in the art a kind of like this common recognition: the expressed enzyme of same genotype can represent the metabolic activity to other substrate medicine to the metabolic activity of specific substrate.Thereby, according to the expressed enzyme of a certain genotype to specific substrate metabolic activity data can analogize the expressed enzyme of this genotype to the metabolic activity of other substrate medicine (as, the metabolic activity of the metabolic activity of the expressed enzyme of this genotype and the expressed enzyme of wild-type can be compared).
In the present embodiment, according to above-mentioned mutational site, wild-type CYP2D6 (* 1) gene of take is template, rite-directed mutagenesis the Nucleotide (becoming C from T) of the 425th of CYP2D6 gene coding region, the expression vector of construction expression saltant type CYP2D6 albumen (called after L142S or CYP2D6.89), after transfection 293FT cell, add CYP2D6 specific probe substrate---Dextromethorphane Hbr and bufuralol, after hatching, by analyzing substrate and meta-bolites amount, detect the In vitro metabolism activity of this saltant type CYP2D6 albumen, with judgement, compare with wild-type CYP2D6.1, whether its enzymatic metabolic activity changes.This experimental design be similar to we at CYP2C9 new mutant body medicine in-vitro the operation steps during for activation analysis, its science is by the most authoritative magazine Pharmacogenomics J approval (document 9 sees reference) of International Pharmaceutical genomics field.
1) vivoexpression of CYP2D6 varient
The plasmid vector (buying from Thermo Scientific company) that comprises wild-type CYP2D6 (* 1) full-length cDNA of take is template, utilizes side-directed mutagenesis to obtain respectively the cDNA of CYP2D6*2, CYP2D6*10 and L142S mutant of the present invention.Side-directed mutagenesis is techniques well known, and those skilled in the art, according to definite template and target, can know beyond all doubtly and how complete this step.By each goal gene that sequence is correct after testing and reference gene Gluc (a kind of secretor type luciferase, its translation product can be secreted in substratum, and fluorescent signal detected by particular agent box; Its skeleton carrier is pIRES pGluc-Basic, purchased from NEB company, article No. N8082S) be connected to respectively double gene expression vector pIRES (purchased from Clontech company, article No. 631605) in A, B multiple clone site, a kind of goal gene and reference gene are positioned under same CMV promotor control, finally obtain double gene expression vector pIRES-Gluc-2D6 (Fig. 2 is shown in by structural representation collection of illustrative plates).Build four kinds of CYP2D6 double gene expression vectors of the cDNA that comprises respectively CYP2D6 (* 1) cDNA, CYP2D6*2cDNA, CYP2D6*10cDNA and L142S mutant of the present invention.
By 5 * 10
5individual 293FT cell (people's renal epithelial cell source, purchased from Invitrogen company) is evenly laid on 6 orifice plates; After incubated overnight, utilize liposome lip2000 (Invitrogen company) transfection 2 μ g plasmid vector pIRES-Gluc-2D6, to express various target protein CYP2D6 and internal reference Protein G luc.After transfection 24h, carry out western hybridization check, determine above-mentioned four kinds of target proteins correction.
Cell cultures and liposome transfection are techniques well known, and the illustration method providing with reference to Invitrogen company just can carry out.
2) viable cell incubated in vitro probe medicament
Continue to cultivate after 24 hours, by single hole cell dissociation in 6 orifice plates resuspended to 300 μ l substratum (use EP pipe carries out).Use the classical probe medicine Dextromethorphane Hbrs of two kinds of CYP2D6 (buying from U.S. Sigma company) and bufuralol (purchase from Canadian Toronto Research Chemicals company) to detect the activity of this enzyme, the final concentration of two kinds of medicines is respectively 20 μ M and 10 μ M.At 37 ℃, at 5%CO
2in incubator, 3h is hatched in 300rpm concussion.From CO
2in incubator, take out culture, add the 0.1MNaOH of 20 μ L and shake vortex 1min that (reason is that bufuralol and Dextromethorphane Hbr are all weak base, solution, with existing with molecular conformation after NaOH alkalization, is extracted out thereby easily enter organic phase in next step extraction process).Add 800 μ L glacial acetic acid ethyl esters, concussion vortex 2min is placed on-40 ℃ of refrigerator 30min, until lower floor is freezing.Take out sample, at 4 ℃, with the centrifugal 10min of 12000g.Upper strata ethyl acetate is transferred in new EP pipe, on 37 ℃ of Nitrogen evaporators, ethyl acetate is dried up.Add 200 μ L separately initial flow phase solution redissolve, after concussion vortex 1min with the centrifugal 5min of 12000g.Supernatant is transferred in sample injection bottle after using initial flow phase solution by dilution in 1: 10.
3) LC-MS/MS detects
After abstraction purification, sample carries out LC-MS/MS detection on 1260-6410 type instrument (U.S. Agilent company), and HPLC adopts ZORBAX SB-C18 post (150mm * 4.6mm, diameter 5 μ m).
Dextromethorphane Hbr testing conditions:
Chromatographic condition
Column temperature: 30 ℃
Chromatographic column: ZORBAX SB-C18 (Agilent, 5 μ m, 4.6 * 150mm)
Sampling volume: 2 μ L
Flow velocity: 0.6mL/min
Working time: 8min
Table 3: Dextromethorphane Hbr mobile phase ratio
| Time (min) | 10mM ammonium acetate (%, v/v) | Methyl alcohol (%, v/v) |
| 0.01 | 40 | 60 |
| 0.5 | 5 | 95 |
| 4 | 5 | 95 |
| 4.01 | 40 | 60 |
| 8 | 40 | 60 |
Mass spectrum condition:
Electron spray(ES) (ESI) ion source (positive ion), many reaction detection scanning (MRM) patterns;
Ionogenic temperature (TEM): 300 ℃;
Curtain gas speed: 11L/min;
Capillary voltage: 4000V;
Dextromethorphane Hbr Q1/Q3:272.2/213.1; 272.2/147.1;
Impact energy=30;
Cracked voltage=140 of parent ion;
Demethyl Dextromethorphane Hbr Q1/Q3:258.2-157.1; 258.2-133.1;
Impact energy=35;
Cracked voltage=135 of parent ion.
Bufuralol testing conditions:
Chromatographic condition:
Column temperature: 30 ℃
Chromatographic column: ZORBAX SB-C18 (Agilent, 5 μ m, 4.6 * 150mm)
Sampling volume: 1 μ L
Flow velocity: 0.6mL/min
Working time: 10min
Table 4: bufuralol mobile phase ratio
| Time (min) | 10mM ammonium acetate (%, v/v) | Methyl alcohol (%, v/v) |
| 0.01 | 30 | 70 |
| 0.8 | 5 | 95 |
| 3 | 5 | 95 |
| 4.01 | 30 | 70 |
| 10 | 30 | 70 |
Mass spectrum condition:
Electron spray(ES) (ESI) ion source (positive ion), many reaction detection scanning (MRM) patterns;
Ionogenic temperature (TEM): 350 ℃;
Curtain gas speed: 11L/min;
Capillary voltage: 4000V;
Bufuralol Q1/Q3:262.2/188.1; 268.2/186.1;
Impact energy=10;
Cracked voltage=110 of parent ion;
1 '-hydroxyl bufuralol Q1/Q3:278.2/186.1; 278.2/159.1;
Impact energy=15;
Cracked voltage=90 of parent ion.
4) LC-MS/MS detects data analysis
2500,1250,500,250,100,20ng/mL prepare Dextromethorphane Hbr, bufuralol and corresponding meta-bolites demethyl Dextromethorphane Hbr, the 1 '-hydroxyl bufuralol (Dextromethorphane Hbr: of different gradient concentrations; 1500,750,300,150,50,25ng/mL demethyl Dextromethorphane Hbr:; 1000,500,250,100,50,25ng/mL bufuralol:; 1 '-hydroxyl bufuralol: 800,400,200,100,50,25ng/mL), after utilizing LC-MS to detect, generate typical curve, and in order to detect the metabolism situation of various CYP2D6 albumen to Dextromethorphane Hbr and bufuralol, then use [product/(product+substrate)] to represent the metabolic rate of this probe medicine, after internal reference Gluc data calibration, with the ratio of the metabolic rate of saltant type and the metabolic rate of wild-type, represent the enzymic activity of mutein.Every kind of albumen repeats respectively 4 times for the experiment of two kinds of medicines, after averaging statistics respectively in Table 5, table 6 and Fig. 3; Various albumen to the typical mass spectrometric detection of Dextromethorphane Hbr and bufuralol the results are shown in Figure 4, Fig. 5.The metabolic capacity of CYP2D6 varient is stronger, the peak area at substrate (Dextromethorphane Hbr or bufuralol) peak will be less, and the peak area at product (demethyl Dextromethorphane Hbr or 1 '-hydroxyl bufuralol) peak will be larger, as can be seen from the figure, new varient CYP2D6.89 obviously reduces compared with wild-type for the metabolic capacity of Dextromethorphane Hbr, be less than typical mutant CYP2D6.2, but be greater than again typical mutant CYP2D6.10 simultaneously; Metabolic capacity for bufuralol is all significantly less than all typical mutant.
Table 5:293FT cell is hatched the enzymic activity result of probe medicament Dextromethorphane Hbr
*refer to the relative value with wild-type CYP2D6.1
*p value < 0.05
Table 6:293FT cell is hatched the enzymic activity result of probe medicament bufuralol
*refer to the relative value with wild-type CYP2D6.1
*p value < 0.05
Detected result shows, with respect to wild-type CYP2D6.1 type, known typical deficient mutants CYP2D6.2 declines not obviously (5% left and right) to the metabolic activity of Dextromethorphane Hbr, but the metabolic activity of another probe medicament bufuralol is obviously declined, and fall is about 16%; Another typical defect type mutant CYP2D6.10 is for all obviously decline (reduction amplitude is higher than 90%) of metabolic activity of two kinds of probe medicaments, this result and existing document are basically identical, show that the data of our vitro detection system acquisition have very high confidence level (referring to reference 7,8).
Utilize this vitro detection system to find out: L142S mutant is respectively 24.82% and 5.90% of wild-type to the metabolic activity of probe medicament Dextromethorphane Hbr and bufuralol.Statistical analysis shows, compares with wild-type, and the metabolic activity of L142S mutant declines, and has significant difference, and the metabolic activity of pointing out this sudden change can cause expressed enzyme obviously reduces.Therefore, in practice, need to consider suitably to regulate on dosage carrying this genotypic individuality, as reduced the usage quantity and the generation of avoiding adverse drug reaction of medicine.This medicine adjustment by gene targeting is even more important for the larger medicine of individual difference (as imipramine, amitriptyline etc.).
Reference
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3.ZHOU?SF.Polymorphism?of?Human?Cytochrome?P4502D6and?Its?Clinical?Significance?Part?II.Clinical?pharmacokinetics.2009,48(12)：761-804.
4. Xu Yan is tender, Gong Sen, and Ji Hongyan, waits .CYP2D6 gene pleiomorphism and clinical meaning thereof. medical Leader .2012,31 (10): 1337-1340.
5.Hersberger?M,Marti-Jaun?J,Rentsch?K,Hanseler?E.Rapid?detection?of?the?CYP2D6
*3,CYP2D6
*4,and?CYP2D6
*6alleles?by?tetra-primer?PCR?and?of?the?CYP2D6
*5allele?by?multiplex?long?PCR.Clin?Chem.2000,46：1072-1077.
6.Lovlie?R,Daly?AK,Molven?A,Idle?JR,Steen?VM.Ultrarapid?metabolizers?of?debrisoquine：characterization?and?PCR-based?detection?of?alleles?with?duplication?of?the?CYP2D6gene.FEBS?Lett.1996,392：30-34.
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*29allele?commonly?present?in?a?black?Tanzanian?population?causing?reduced?catalytic?activity.Pharmacogenetics2001,11：417-427.
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Sequence:
SEQ ID NO.1: allelotrope sequence
ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGCAGAGGTGCTGAGGCTCCCCTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCAGAAGCCAGGCTGAGAAGGGGAAGCAGGTTTGGGGGACGTCCTGGAGAAGGGCATTTATACATGGCATGAAGGACTGGATTTTCCAAAGGCCAAGGAAGAGTAGGGCAAGGGCCTGGAGGTGGAGCTGGACTTGGCAGTGGGCATGCAAGCCCATTGGGCAACATATGTTATGGAGTACAAAGTCCCTTCTGCTGACACCAGAAGGAAAGGCCTTGGGAATGGAAGATGAGTTAGTCCTGAGTGCCGTTTAAATCACGAAATCGAGGATGAAGGGGGTGCAGTGACCCGGTTCAAACCTTTTGCACTGTGGGTCCTCGGGCCTCACTGCCTCACCGGCATGGACCATCATCTGGGAATGGGATGCTAACTGGGGCCTCTCGGCAATTTTGGTGACTCTTGCAAGGTCATACCTGGGTGACGCATCCAAACTGAGTTCCTCCATCACAGAAGGTGTGACCCCCACCCCCGCCCCACGATCAGGAGGCTGGGTCTCCTCCTTCCACCTGCTCACTCCTGGTAGCCCCGGGGGTCGTCCAAGGTTCAAATAGGACTAGGACCTGTAGTCTGGGGTGATCCTGGCTTGACAAGAGGCCCTGACCCTCCCTCTGCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCCTGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGCAAGCAGCGGTGGGGACAGAGACAGATTTCCGTGGGACCCGGGTGGGTGATGACCGTAGTCCGAGCTGGGCAGAGAGGGCGCGGGGTCGTGGACATGAAACAGGCCAGCGAGTGGGGACAGCGGGCCAAGAAACCACCTGCACTAGGGAGGTGTGAGCATGGGGACGAGGGCGGGGCTTGTGACGAGTGGGCGGGGCCACTGCCGAGACCTGGCAGGAGCCCAATGGGTGAGCGTGGCGCATTTCCCAGCTGGAATCCGGTGTCGAAGTGGGGGCGGGGACCGCACCTGTGCTGTAAGCTCAGTGTGGGTGGCGCGGGGCCCGCGGGGTCTTCCCTGAGTGCAAAGGCGGTCAGGGTGGGCAGAGACGAGGTGGGGCAAAGCCTGCCCCAGCCAAGGGAGCAAGGTGGATGCACAAAGAGTGGGCCCTGTGACCAGCTGGACAGAGCCAGGGACTGCGGGAGACCAGGGGGAGCATAGGGTTGGAGTGGGTGGTGGATGGTGGGGCTAATGCCTTCATGGCCACGCGCACGTGCCCGTCCCACCCCCAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGCGCGAGCAGA?GGCGCTTCTCCGTGTCCACCTTGCGCAACTCGGGCCTGGGCAAGAAGTCGCTGGAGCAGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTCGCCAACCACTCCGGTGGGTGATGGGCAGAAGGGCACAAAGCGGGAACTGGGAAGGCGGGGGACGGGGAAGGCGACCCCTTACCCGCATCTCCCACCCCCAGGACGCCCCTTTCGCCCCAACGGTCTCTTGGACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCGGAGCGAGAGACCGAGGAGTCTCTGCAGGGCGAGCTCCCGAGAGGTGCCGGGGCTGGACTGGGGCCTCGGAAGAGCAGGATTTGCATAGATGGGTTTGGGAAAGGACATTCCAGGAGACCCCACTGTAAGAAGGGCCTGGAGGAGGAGGGGACATCTCAGACATGGTCGTGGGAGAGGTGTGCCCGGGTCAGGGGGCACCAGGAGAGGCCAAGGACTCTGTACCTCCTATCCACGTCAGAGATTTCGATTTTAGGTTTCTCCTCTGGGCAAGGAGAGAGGGTGGAGGCTGGCACTTGGGGAGGGACTTGGTGAGGTCAGTGGTAAGGACAGGCAGGCCCTGGGTCTACCTGGAGATGGCTGGGGCCTGAGACTTGTCCAGGTGAACGCAGAGCACAGGAGGGATTGAGACCCCGTTCTGTCTGGTGTAGGTGCTGAATGCTGTCCCCGTCCTCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGACCCAGCTGGATGAGCTGCTAACTGAGCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGTGAGAGTGGCTGCCACGGTGGGGGGCAAGGGTGGTGGGTTGAGCGTCCCAGGAGGAATGAGGGGAGGCTGGGCAAAAGGTTGGACCAGTGCATCACCCGGCGAGCCGCATCTGGGCTGACAGGTGCAGAATTGGAGGTCATTTGGGGGCTACCCCGTTCTGTCCCGAGTATGCTCTCGGCCCTGCTCAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTGCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGTGAGCCCATCTGGGAAACAGTGCAGGGGCCGAGGGAGGAAGGGTACAGGCGGGGGCCCATGAACTTTGCTGGGACACCCGGGGCTCCAAGCACAGGCTTGACCAGGATCCTGTAAGCCTGACCTCCTCCAACATAGGAGGCAAGAAGGAGTGTCAGGGCCGGACCCCCTGGGTGCTGACCCATTGTGGGGACGCATGTCTGTCCAGGCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGATTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGTGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGTAGGCCTGGCGCCCTCCTCACCCCAGCTCAGCACCAGCACCTGGTGATAGCCCCAGCATGGCTACTGCCAGGTGGGCCCACTCTAGGAACCCTGGCCACCTAGTCCTCAATGCCACCACACTGACTGTCCCCACTTGGGTGGGGGGTCCAGAGTATAGGCAGGGCTGGCCTGTCCATCCAGAGCCCCCGTCTAGTGGGGAGACAAACCAGGACCTGCCAGAATGTTGGAGGACCCAACGCCTGCAGGGAGAGGGGGCAGTGTGGGTGCCTCTGAGAGGTGTGACTGCGCCCTGCTGTGGGGTCGGAGAGGGTACTGTGGAGCTTCTCGGGCGCAGGACTAGTTGACAGAGTCCAG?CTGTGTGCCAGGCAGTGTGTGTCCCCCGTGTGTTTGGTGGCAGGGGTCCCAGCATCCTAGAGTCCAGTCCCCACTCTCACCCTGCATCTCCTGCCCAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCTCAGCAGGTGCCTGTGGGGAGCCCGGCTCCCTGTCCCCTTCCGTGGAGTCTTGCAGGGGTATCACCCAGGAGCCAGGCTCACTGACGCCCCTCCCCTCCCCACAGGCCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAGCACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAG
SEQ ID NO.2: encoding sequence
ATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGTTGCGGCGCCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCCTGTGCCCATCACCCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGCGCGAGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTCGGGCCTGGGCAAGAAGTCGCTGGAGCAGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTCGCCAACCACTCCGGACGCCCCTTTCGCCCCAACGGTCTCTTGGACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCTGAATGCTGTCCCCGTCCTCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAAAAGGCTTTCCTGACCCAGCTGGATGAGCTGCTAACTGAGCACAGGATGACCTGGGACCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTGCGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGATTCATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGTGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGGAACGACACTCATCACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCTCAGCAGGCCGCCGTGCATGCCTCGGGGAGCCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAGCACTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCG?CTAG
SEQ ID NO.3: protein sequence
MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYPPGPLPLPGLGNLLHVDFQNTPYCFDQLRRRFGDVFSLQLAWTPVVVLNGLAAVREALVTHGEDTADRPPVPITQILGFGPRSQGVFLARYGPAWREQRRFSVSTLRNSGLGKKSLEQWVTEEAACLCAAFANHSGRPFRPNGLLDKAVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLLHIPALAGKVLRFQKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDENLRIVVADLFSAGMVTTSTTLAWGLLLMILHPDVQRRVQQEIDDVIGQVRRPEMGDQAHMPYTTAVIHEVQRFGDIVPLGVTHMTSRDIEVQGFRIPKGTTLITNLSSVLKDEAVWEKPFRFHPEHFLDAQGHFVKPEAFLPFSAGRRACLGEPLARMELFLFFTSLLQHFSFSVPTGQPRPSHHGVFAFLVSPSPYELCAVPR
SEQ ID NO.14: nucleic acid fragment
GCGCAACTCGGGCCTG
SEQ ID NO.15: nucleic acid fragment
CTTGCGCAACTCGGGCCTG
SEQ ID NO.16: nucleic acid fragment
CACCTTGCGCAACTCGGGCCTGGGCAAG
SEQ ID NO.17: nucleic acid fragment
GTGTCCACCTTGCGCAACTCGGGCCTGGGCAAGAAGTC
SEQ ID NO.18: nucleic acid fragment
CTTCTCCGTGTCCACCTTGCGCAACTCGGGCCTGGGCAAGAAGTCGCTGG
SEQ ID NO.19: oligonucleotide fragment
CTTGCCCAGGCCCGAG
SEQ ID NO.20: oligonucleotide fragment
CTTCTTGCCCAGGCCCGAGTTG
SEQ ID NO.21: oligonucleotide fragment
CCAGGCCCGAGTTGC
SEQ ID NO.22: oligonucleotide fragment
GACTTCTTGCCCAGGCCCGAG
SEQ ID NO.23: oligonucleotide fragment
CAGCGACTTCTTGCCCAGGCCCGAGTTG
Claims (10)
1. nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1678th corresponding to SEQ ID NO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, and wherein the Nucleotide of the 1678th is C; Or described nucleic acid fragment comprises the mutational site of the 425th corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 425th is C; Or be the reverse complementary sequence of above-mentioned nucleic acid fragment.
2. nucleic acid fragment according to claim 1, is characterized in that, the length of described nucleic acid fragment is 10-100,101-200, a 201-500 or 501-1000 Nucleotide; Preferably, the length of described nucleic acid fragment is 10-20,21-30,31-40,41-50,51-60, a 61-100 or 101-300 Nucleotide; Further preferably, described nucleic acid fragment be SEQ ID NO.1,2 or 14-18 shown in sequence.
3. allele specific oligonucleotide, described oligonucleotide with contain corresponding to the 1678th of SEQ ID NO.1 or corresponding to allelotrope fragment or all or part of hybridization of its reverse complementary sequence in the mutational site of the 425th of SEQ ID NO.2, wherein the Nucleotide in the mutational site of the 425th of the 1678th of SEQ ID NO.1 the or SEQ IDNO.2 is C; Described allelotrope fragment is at least 10 continuous nucleotides or its reverse complementary sequence in the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
4. allele specific oligonucleotide according to claim 3, is characterized in that, described oligonucleotide is probe or primer; Preferably, when described oligonucleotide is probe, the length of described oligonucleotide is 5-100 Nucleotide; When described oligonucleotide is primer, the length of described oligonucleotide is 15-40 Nucleotide; Preferably, when described oligonucleotide is probe, described mutational site is positioned at center or about center of probe sequence; When described oligonucleotide is primer, described mutational site is positioned at 3 ' end of primer.
5. allele specific oligonucleotide according to claim 4, is characterized in that, described oligonucleotide is the sequence as shown in SEQ ID NO.19-23.
One kind for detection of and/or the test kit of analysis list base mutation, comprise nucleic acid fragment according to claim 1 and 2 or according to the allele specific oligonucleotide described in claim 3-5 any one, or comprise can be as single base mutation described in primer amplification but do not comprise the nucleic acid fragment of this list base; Described single base is corresponding to the 1678th or the 425th of SEQ ID NO.2 of SEQ ID NO.1; Preferably, described test kit comprises the sequence fragment shown in SEQ ID NO.4 and/or SEQ ID NO.5 and/or SEQ ID NO.10.
7. nucleic acid fragment according to claim 1 and 2 or the application in detecting CYP2D6 transgenation according to the allele specific oligonucleotide described in claim 3-5 any one, wherein said nucleic acid fragment or oligonucleotide are as probe or primer; Or the application in the medicine of preparation detection CYP2D6 transgenation; Or as the application that detects the check mark thing of CYP2D6 transgenation.
8. a medication guide, comprises the 1678th or the base of the 425th of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2D6 gene in testing sample, when the base in described site is C, adjusts the dosage through the medicine of CYP2D6 metabolism.
9. a method for analysis of nucleic acids, described method comprise analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the Nucleotide of the 1678th or analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the Nucleotide of the 425th; Preferably, described method is sequencing, restriction fragment length polymorphism analysis or probe hybridization method.
10.CYP2D6 albumen or its fragment or varient, described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the Serine of the 142nd corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3.
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| CN101434994A (en) * | 2008-12-25 | 2009-05-20 | 上海交通大学 | Method for detecting CYP2C9 gene exon 9 mononucleotide polymorphism |
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